4.9. Calcein Leakage Assay

Calcein-entrapped LUVs composed of egg yolk phosphatidylethanolamine (EYPE)/egg yolk phosphatidylglycerol EYPG (7:3, w/w) were prepared by vortexing the dried lipid in dye buffer solution (70 mM calcein, 10 mM Tris, 150 mM NaCl, and 0.1 mM Ethylenediaminetetraacetic acid (EDTA), pH 7.4) as described previously. The leakage of calcein from the LUVs was monitored by measuring fluorescence intensity at an excitation wavelength of 490 nm and an emission wavelength of 520 nm on a model RF-5301PC spectrophotometer (Shimadzu, Kyoto, Japan). For determination of 100% dye-release, 10% Triton-X100 in Tris-buffer (20 μL) was added to dissolve the vesicles. The experiment was performed in triplicate, and the percentage of dye-leakage caused by the peptides was calculated as follows: Dye-leakage (%) = 100 × (F − F₀)/(F_t − F₀), where F is the fluorescence intensity shown by the peptides and where F₀ and F_t are fluorescence intensities without the peptides and with Triton X-100, respectively.